We propose to advance a systems biology framework for an integrative understanding of the signaling and transcriptional events in early vertebrate embryonic development leading to major ectodermal, mesodermal, and endodermal cell types, using the Xenopus laevis model system. Controlled manipulation of three signaling pathways, Bmp, Nodal, and Wnt, will be used to drive multipotent embryonic cells in different well-defined developmental directions. We will characterize each of the endpoint cell types and many intermediates, providing a step- by-step analysis of the changes in protein and mRNA levels as a cell makes fate choices. Our team is a close collaboration between two expert embryologists/cell biologists (Kirschner and Gerhart) with an expert in systematic analysis and statistical inference (Peshkin) and an expert in proteomic mass spectrometry (Gygi). Our recently developed methods will allow us to reach unprecedented levels of detection and quantification of proteins, protein post-translational modifications, and mRNA transcripts. The large size of Xenopus eggs and embryos will also permit single-cell studies in some cases. We hypothesize that high quality proteomic profiling of early development will produce new key insights into developmental signaling pathways. Our dataset will provide an unprecedented degree of systems-level knowledge on the localization- and time-dependent actions of proteins in early vertebrate development, in cells fated to become different cell types. This information will be new for many biological problems, and revolutionary for embryology. Our suite of methods will allow rapid profiling of the effect of developmental signals on key pathways, and on vertebrate embryonic development in general. These key pathways are also important in later developmental processes, in childhood development, tissue and cell turnover in adults, and in approaches to regenerative medicine based on stem cell biology. They are also largely conserved across species, promising rapid transfer from the model system to human patients. We expect that these data will inform on heritable defects in human embryos and children in new ways.